Authors: Hu Lili, Ai Ming, Yang Hongxia, Jiang Shuanghong
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Studies show that retinal neurodegeneration may precede retinal microvascular changes in diabetes mellitus.The apoptosis of retinal ganglion cells (RGCs) is an early finding in retinal neurodegeneration.Toll-like receptor 4 (TLR4) is proved to be up-regulated in diabetic rats retina.However, the impact of TLR4 on RGCs damage in retinal neurodegeneration is poorly understood.
The aim of this study was to investigate the expressing change of TLR4 induced by high glucose in RGCs in order to offer a basis for the prevention diabetic retinal neurodegeneration and the study on targeting drugs.
RGCs were isolated and purified from the retinas of SPF SD rats aged postnatal 1-3 days by using papain digestion method and then were identified by immunofluorescence technology to detect the expression of Brn3a, a specific marker of RGCs.The cells were divided into normal control group and 10, 20, 30 mmol/L glucose groups.The expressions of TLR4 mRNA and protein in the cells were detected by real-time fluorescence quantitative PCR and Western blot analysis in 24 and 48 hours after addtion of glucose.All procedures performed in studies were in accordance with the Association for National Institutes of Health (NIH) Statement for the Care and Use of Laboratory Animals recommendations.The protocol was approved by the Ethics Committee of Renmin Hospital of Wuhan University.Every effort was made to minimize animal discomfort and stress.
The normal cells grew well with the shape of near roundness after inoculaton.The cells were gradually enlarged and clustered with obvious axons and dendrites 24 hours after purifying.Brn3a showed the positive expression in cultured cells.At 24 hours and 48 hours after glucose culture, the cell structures were gradually invisible in most cells.The expressions of TLR4 mRNA in the cells were 0.945±0.237, 1.180±0.193 and 0.827±0.213 at 24 hours and 1.509±0.422, 2.433±0.617 and 1.435±0.410 at 48 hours after culture in the 10, 20 and 30 mmol/L glucose groups, respectively, which were significantly higher than 0.600±0.099 and 0.724±0.302 in the normal control group (all at P<0.01). The expressions of TLR4 protein in the cells were 0.442±0.147, 0.626±0.128 and 0.330±0.153 at 24 hours and 0.464±0.121, 0.930±0.441 and 0.394±0.158 at 48 hours after culture in the 10, 20 and 30 mmol/L glucose groups, respectively, which were significantly higher than 0.090±0.050 and 0.094±0.070 in the normal control group (all at P<0.01).
A large number of RGCs die in a high-glucose environment in vitro, meanwhile, the expression of TLR4 up-regulates in the cells, indicating that TLR4 maybe participate in the damage of RGCs induced by high glucose.